Dr Sheena McGowan
Head, Structural Microbiology Laboratory
Department of Biochemistry & Molecular Biology
Tel: +61 3 9902 9309 (Office)
Facsimile: +61 3 9902 9500
Building 77, Level 2, Room 243
Victoria 3800, Australia
About the Structural Microbiology Laboratory
The McGowan laboratory is interested in characterising novel molecular drug targets. The growing problem of drug resistance within the microbial world underlies the critical need to develop new treatments to prevent and control resistant infections. Therefore, the development of new therapeutic approaches for the treatment of microbial infections is a critically important goal. Our lab has a strong research focus in the design of novel anti-malarial drugs as well as other parasitic and bacterial diseases. Primarily we are a structural biology laboratory using techniques in X-ray crystallography, biochemistry and biophysics to analyse our proteins of interest. We use this mechanistic information to design inhibitors or analogues with potential applications in human medicine.
New drugs for malaria
Malaria is the world's most prevalent parasitic disease, with over 200 million cases and 627,000 resultant deaths annually. Nearly 1300 children under the age of 5 die everyday from malaria. Alarmingly, the spread of drug-resistant parasites has rendered most antimalarials ineffective and has made the discovery of new therapeutics a global health priority.
Development of phage lysins as antibacterial agents
Bacteriophage lysins are proteins that act to kill bacteria by digesting their cell wall. This makes them attractive as novel antibacterial agents.
Komagal Kannan Sivaraman
Our laboratory is located in the Science and Technology Research Innovation Precinct on the Clayton Campus (Building 77, 23 Innovation Walk) and has access to cutting edge infrastructure including the Macromolecular Crystallisation Facility, Micromon DNA sequencing and Protein Production Unit. We are located < 1 km from the Australian Synchrotron.
Antimalarial Research Program
Despite one century of control and eradication campaigns, malaria remains one of the world's most devastating diseases. Our once powerful therapeutic weapons are losing the war against the Plasmodium parasite, whose ability to rapidly develop and spread drug resistance hamper past and present malaria control efforts. Finding new and effective treatments for malaria is now a top global health priority, fuelling an increase in funding and promoting open source collaborations between researchers and pharmaceutical consortia around the world. The result of this is rapid advances in drug discovery approaches and technologies. Structure-based drug discovery (SBDD) is a foundation for many antimalarial drug development programs and has been invaluable to the development of a number of current pre-clinical and clinical candidates.
The McGowan laboratory has a strong focus on protease biology and the development of protease inhibitors for use in human medicine.
Some specific projects are detailed below.
1. Metalloaminopeptidases as drug targets
The Pf malaria parasite employs metalloaminopeptidase enzymes (PfMAPS) that are required for parasite survival. These enzymes are attractive drug targets. Agents that inhibit the activity of these enzymes thus represent leads for the development of new antimalarial drugs. To date, nine PfMAPs have been identified. Four of these are methionine aminopeptidases. The other five enzymes comprise a prolyl iminopeptidase (or post- prolyl aminopeptidase), a prolyl aminopeptidase, a M17 leucine aminopeptidase, an M1 alanine aminopeptidase and an M18 aspartyl aminopeptidase. Recent studies show that the PfMAPs have roles in hemoglobin digestion, housekeeping functions and in conjunction with the parasite proteosome. The essential nature of the PfMAP activity has sparked interest in using them as targets for the development of novel antimalarials. To date, our research into the M1, M17 and M18 PfMAPs have validated these enzymes as antimalarial drug targets. They are the main focus of our research.
Protease enzymes are responsible for breaking down human haemoglobin and providing food for the parasite. Inhibition of protease activity starves the parasite.
Determine the regulation and control of proteolysis of the PfMAPs
Aim: To use molecular and computational biology, biochemistry and protein crystallography to identify how substrates access the buried active sites of the PfMAPs.
Successful drug design campaigns attempt to mimic the natural substrate as closely as possible. To mimic and imitate the natural substrate of enzymes, it is vital that we understand how the substrate accesses the active site. The complex quarternary structures of M1, M17 and M18 act to control proteolysis. Their assembly and flexibility control peptide substrate access to the active site but also act to remove the free amino acid proteolysis product. Without this control, biological activity would be compromised. Identifying the entry and exit points of the enzymes, would allow the future design of a novel class of aminopeptidase inhibitors - entry blockers. Further, the knowledge we will gain from these studies will be vital to achieve selectivity of our compounds, targeting the compound to parasite enzyme rather than its mammalian homolog.
We will use our atomic data and knowledge of the M1, M17 and M18 enzymes will guide a detailed mutagenesis study to map entry / exit 'hotspots' by which peptides can access the inner active site cavities of the enzymes and generate a model of product release. Further, we will undertake a computational biology approach to consider the flexibility and dynamics of each enzyme. Molecular dynamic simulations offer the ability to calculate the behaviour of a molecular system over time, extending the information gained from static X-ray crystallography. Computational approaches, coupled closely with experimental validation, provides a useful tool for further understanding protein structure and protein-ligand interactions.
Development novel PfMAP inhibitors as anti-malarial agents
Aim: Use rational drug design, structure-activity studies and SBDD to develop novel inhibitors that target either single or multiple PfMAP enzymes.
As with all drug development for new and novel targets, multiple fronts and varied approaches are essential for ultimate success. For example, the influenza drugs Relenza and Tamiflu were independently developed from SBDD initiatives. Both drugs have had a significant impact on public health. They inhibit the same target, treat the same disease but vary in their oral bioavailability, size effects and susceptibility to resistance. Currently PfMAP inhibitors are designed to chelate the essential metal ion in the active sites of each of the enzymes. Recently work from my laboratory and others have described phosphonopeptides, peptide-based bestatin analogues, and hydroxamate derivatives as potential single-enzyme inhibitors. An orally bioavailable hydroxamate-containing ester, CHR-2863, was also shown to be efficacious against murine malaria. In collaboration with Prof. Peter Scammells (MIPS) these agents will continue to be developed during the next five years. We will use a combination of medicinal chemistry, computational biology, biochemistry, parasitology and X-ray crystallography to continue to elaborate and refine these inhibitors.
Agents targeted to two or more of the PfMAPs are considered to be an exciting way forward in the development of lead compounds. An ideal agent is a compound that would inhibit all three PfMAPs. Agents that are active against multiple targets (a combination therapy) are generally superior as this reduces the likelihood of parasites being able to rapidly evolve resistance. Although the PfMAP super-family is large and divergent, the enzymes utilise a common catalytic mechanism by the coordination of one or two cations in the active site. My research has provided functional and structural information about the precise specificity of the active site binding pockets of the PfMAPs. Using this information, we have recently identified candidate compounds for dual and tri-enzyme inhibitors. We will elaborate these compounds with physicochemical properties appropriate for an oral drug.
2. Develop compounds targeting the Falcipains
Aim: Develop pre-clinical candidates / lead compounds targeting the food vacuole falcipains
The erythrocytic stages of Pf rely on hemoglobin degradation as the principal source of amino acids for protein synthesis. Hemoglobin is transported to an acidic food vacuole where it is metabolised by a series of proteases. Importantly, this function is carried out by entirely different classes of proteases in vertebrates, thus making parasitic proteases attractive drug targets. The falcipains (FPs) are a recently identified and characterised family of papain-like cysteine proteases, comprised of four members - FP1, FP2, FP2' and FP3. Inhibiting key enzymes in the hemoglobin degradation pathway leads to interruption of the nutrition source. FP inhibition therefore eliminates the parasite by starvation, making the food vacuole FPs an attractive target for the development of new drugs targeting malaria via a novel mode of action. Gallinamide A is a recently isolated, potent antiplasmodial compound that exerts its anti-parasitic activity by potently inhibiting the food vacuole FPs. In collaboration with A/Prof Richard Payne (University of Sydney) we will characterise novel gallinamide A derivatives that have been designed for biological activity as well as pharmacokinetic profile. A/Prof Payne has pioneered an innovative synthetic methodology to rapidly assemble potent and selective inhibitors of gallinamide A. A key to the success of this project is detailed atomic information that is provided in parallel with in vitro and in vivo assessment. My laboratory will provide this pivotal contribution to the project.
3. Develop a new class of inhibitors against AMA1
Aim: Fragment-based development of small molecule inhibitors of the AMA1-RON2 interaction
Apical membrane antigen 1 (AMA1) plays an essential role in the invasion of host cells by malaria parasites, forming a complex with parasite rhoptry neck (RON) proteins as part of a moving junction that develops between the host cell and the invading parasite. Agents that inhibit this interaction prevent host cell invasion and thus represent leads for the development of drugs that would block host cell invasion by malarial parasites. In collaboration with Prof Ray Norton (MIPS), we have an extended MIPS research team identifying small molecule compounds that can inhibit the AMA1-RON2 complex. In this project, we are using Fragment-based drug discovery (FBDD). Fragment-based drug discovery (FBDD) involves identifying small, low-affinity ligands (‘fragments') and combining these to produce larger, higher-affinity ligands. Fragment-based approaches are being used increasingly in the pharmaceutical industry, particularly against challenging targets such as protein-protein interaction surfaces, many of which had been considered previously to be undruggable. Fragment-based screening provides more ‘developable' compounds than traditional drug discovery approaches which optimise a high affinity ‘hit'. Most importantly, fragment methods produce lead candidates with physicochemical properties that are likely to result in orally bioavailable compounds.
The target region of the AMA1 protein is a highly mobile hydrophobic pocket that interacts with RON2 protein. To improve our understanding of this dynamic pocket, we are using experimental (NMR, crystallography, biochemistry) and computational (molecular dynamics, docking) biology to provide insight into the conformational changes that the protein undergoes. We are also investigating the strain specificity of AMA1 and use this information to guide the elaboration of compounds. To do this, we will solve the X-ray crystal structures of medically relevant P. falciparum strains.
Phage lysins as New Antimicrobials
The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria.
PlyC is the most potent lysin characterized to date. We have determined its molecular structure, revealing a complicated arrangement of proteins that work together as a bacteriolytic machine. The PlyC architecture has new and novel therapeutic potential. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Engineering this molecule could deliver a multitude of broad antimicrobial agents.
Mistry§, Drinkwater§, Ruggeri, Kannan Sivaraman, Loganathan, Fletcher, Drag, Paiardini, Avery, Scammells & McGowan (2014) "A Two-pronged Attack: Dual Inhibition of M1 and M17 Metalloaminopeptidases by a Novel Series of Hydroxamic acid-based Inhibitors" J. Med. Chem. doi.org/10.1021/jm501323a. § JOINT FIRST AUTHORS.
Lim, Yang, Bankala, Kannan Sivaraman, Chandrashekaran, Kass, MacRaild, Devine, Debono, Anders, Scanlon, Scammells, Norton & McGowan "X-ray crystal structure of Apical Membrane Antigen 1 from Plasmodium falciparum FVO Biochemistry. doi.org/10.1021/bi5012089.
Drinkwater, N. and McGowan, S. (2014) "From crystal to compound: structure-based antimalarial drug discovery". Biochem J In Press (accepted 22 April). FRONT COVER
Wang, MacRaild, Mohanty, Mobli, Cowieson, McGowan, Anders, Simpson, Norton & Scanlon. (2014) "Molecular Insights into the Interaction between Plasmodium falciparum Apical Membrane Antigen 1 and an Invasion-Inhibitory Peptide" PlosONE (Accepted 3/9/14)
Meyer, Aragao, Mudie, Caradoc-Davies, McGowan, Bertling, Groenewegen, Quenette, Bond, Buckle & Androulakis. (2014) "Operation of the Australian Store.Synchrotron for Macromolecular Crystallography" Acta Cryst Section D, Accepted 12/7/14
Gras, Byzia, Gilbert, McGowan, Drag, Silvestre, Niepceron, Lecaille, Lalmanach & Brossier (2014) "Aminopeptidase N1 (Et-ApN1), a M1 metalloprotease of the apicomplexan parasite Eimeria tenella participates in parasite development" Eukaryot Cell 13(7), 884-895.
McGowan, S. (2013) "Working in concert: the metalloaminopeptidases from Plasmodium falciparum". Curr Opin Struct Biol 23, 828-835
McGowan, S. (2013) "Sitagliptin does not inhibit the M1 alanyl aminopeptidase from Plasmodium falciparum". Bioinformation 9, 661-662
Kannan Sivaraman, K., Paiardini, A., Sienczyk, M., Ruggeri, C., Oellig, C. A., Dalton, J. P., Scammells, P. J., Drag, M., and McGowan, S. (2013) "Synthesis and Structure-Activity Relationships of Phosphonic Arginine Mimetics as Inhibitors of the M1 and M17 Aminopeptidases from Plasmodium falciparum". J Med Chem 56, 5213-5217 Citations: 5
Sivaraman, K. K., Oellig, C. A., Huynh, K., Atkinson, S. C., Poreba, M., Perugini, M. A., Trenholme, K. R., Gardiner, D. L., Salvesen, G., Drag, M., Dalton, J. P., Whisstock, J. C., and McGowan, S. (2012) "X-ray crystal structure and specificity of the Plasmodium falciparum malaria aminopeptidase PfM18AAP". J Mol Biol 422, 495-507 JOURNAL COVER ILLUSTRATION
Poreba, M., McGowan, S., Skinner-Adams, T., Trenholme, K. R., Gardiner, D. L., Whisstock, J. C., To, J., Salvesen, G. S., Drag, M., and Dalton, J. P. (2012) "Fingerprinting the substrate specificity of M1 and M17 neutral aminopeptidases of human malaria, Plasmodium falciparum". PloSONE 2, e31938
McGowan, S., Buckle, A. M., Mitchell, M. S., Hoopes, J. T., Gallagher, D. T., Heselpoth, R. D., Shen, Y., Reboul, C. F., Law, R. H., Fischetti, V. A., Whisstock, J. C., and Nelson, D. C. (2012) "X-ray crystal structure of the streptococcal specific phage lysin PlyC". Proc Natl Acad Sci U S A 109, 12752-12757
Velmourougane, G., Harbut, M. B., Dalal, S., McGowan, S., Oellig, C. A., Meinhardt, N., Whisstock, J. C., Klemba, M., and Greenbaum, D. C. (2011) "Synthesis of new (-)-bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase". J Med Chem 54, 1655-1666
Harbut, M. B., Velmourougane, G., Dalal, S., Reiss, G., Whisstock, J. C., Onder, O., Brisson, D., McGowan, S., Klemba, M., and Greenbaum, D. C. (2011) "Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases". Proc Natl Acad Sci U S A 108, E526-534 Citations: 21
Winter, K. L., Isbister, G. K., McGowan, S., Konstantakopoulos, N., Seymour, J. E., and Hodgson, W. C. (2010) "A pharmacological and biochemical examination of the geographical variation of Chironex fleckeri venom". Toxicology letters 192, 419-424
McGowan, S., Oellig, C. A., Birru, W. A., Caradoc-Davies, T. T., Stack, C. M., Lowther, J., Skinner-Adams, T., Mucha, A., Kafarski, P., Grembecka, J., Trenholme, K. R., Buckle, A. M., Gardiner, D. L., Dalton, J. P., and Whisstock, J. C. (2010) "Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases". Proc Natl Acad Sci U S A 107, 2449-2454
Kennan, R. M., Wong, W., Dhungyel, O. P., Han, X., Wong, D., Parker, D., Rosado, C. J., Law, R. H., McGowan, S., Reeve, S. B., Levina, V., Powers, G. A., Pike, R. N., Bottomley, S. P., Smith, A. I., Marsh, I., Whittington, R. J., Whisstock, J. C., Porter, C. J., and Rood, J. I. (2010) "The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates". PLoS pathogens 6, e1001210
Skinner-Adams, T. S., Stack, C. M., Trenholme, K. R., Brown, C. L., Grembecka, J., Lowther, J., Mucha, A., Drag, M., Kafarski, P., McGowan, S., Whisstock, J. C., Gardiner, D. L., and Dalton, J. P. (2010) "Plasmodium falciparum neutral aminopeptidases: new targets for anti-malarials". Trends Biochem Sci 35, 53-61 JOURNAL COVER ILLUSTRATION
Ong, P. C., Golding, S. J., Pearce, M. C., Irving, J. A., Grigoryev, S. A., Pike, D., Langendorf, C. G., Bashtannyk-Puhalovich, T. A., Bottomley, S. P., Whisstock, J. C., Pike, R. N., and McGowan, S. (2009) "Conformational change in the chromatin remodelling protein MENT". PLoS One 4, e4727
McGowan, S., Porter, C. J., Lowther, J., Stack, C. M., Golding, S. J., Skinner-Adams, T. S., Trenholme, K. R., Teuscher, F., Donnelly, S. M., Grembecka, J., Mucha, A., Kafarski, P., Degori, R., Buckle, A. M., Gardiner, D. L., Whisstock, J. C., and Dalton, J. P. (2009) "Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase". Proc Natl Acad Sci U S A 106, 2537-2542
Fischer, K., Langendorf, C. G., Irving, J. A., Reynolds, S., Willis, C., Beckham, S., Law, R. H., Yang, S., Bashtannyk-Puhalovich, T. A., McGowan, S., Whisstock, J. C., Pike, R. N., Kemp, D. J., and Buckle, A. M. (2009) "Structural mechanisms of inactivation in scabies mite serine protease paralogues". J Mol Biol 390, 635-645
Cheung, J. K., Awad, M. M., McGowan, S., and Rood, J. I. (2009) "Functional analysis of the VirSR phosphorelay from Clostridium perfringens". PLoS One 4, e5849
Androulakis, S., Schmidberger, J., Bate, M. A., DeGori, R., Beitz, A., Keong, C., Cameron, B., McGowan, S., Porter, C. J., Harrison, A., Hunter, J., Martin, J. L., Kobe, B., Dobson, R. C., Parker, M. W., Whisstock, J. C., Gray, J., Treloar, A., Groenewegen, D., Dickson, N., and Buckle, A. M. (2008) "Federated repositories of X-ray diffraction images". Acta Crystallogr D Biol Crystallogr D64, 810-814
Porter, C. J., Schuch, R., Pelzek, A. J., Buckle, A. M., McGowan, S., Wilce, M. C., Rossjohn, J., Russell, R., Nelson, D., Fischetti, V. A., and Whisstock, J. C. (2007) "The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis". J Mol Biol 366, 540-550
*Ong, P. C., *McGowan, S., Pearce, M. C., Irving, J. A., Kan, W. T., Grigoryev, S. A., Turk, B., Silverman, G. A., Bottomley, S. P., Whisstock, J. C., and Pike, R. N. (2007) "DNA accelerates the inhibition of human cathepsin V by serpins". J. Biol. Chem. 282, 36980-36986
*Joint first authors.
Law, R. H., Zhang, Q., McGowan, S., Buckle, A. M., Silverman, G. A., Wong, W., Rosado, C. J., Langendorf, C. G., Pike, R. N., Bird, P. I., and Whisstock, J. C. (2006) "An overview of the serpin superfamily". Genome Biol 7, 216
McGowan, S., Buckle, A. M., Irving, J. A., Ong, P. C., Bashtannyk-Puhalovich, T. A., Kan, W. T., Henderson, K. N., Bulynko, Y. A., Popova, E. Y., Smith, A. I., Bottomley, S. P., Rossjohn, J., Grigoryev, S. A., Pike, R. N., and Whisstock, J. C. (2006) "X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation". EMBO J 25, 3144-3155
McGowan, S., O'Connor, J. R., Cheung, J. K., and Rood, J. I. (2003) "The SKHR motif is required for biological function of the VirR response regulator from Clostridium perfringens". J Bacteriol 185, 6205-6208 Citations: 6
McGowan, S., Lucet, I. S., Cheung, J. K., Awad, M. M., Whisstock, J. C., and Rood, J. I. (2002) "The FxRxHrS motif: a conserved region essential for DNA binding of the VirR response regulator from Clostridium perfringens". J Mol Biol 322, 997-1011 Citations: 13
Gardiner, D. L., Dalton, J. P., and McGowan, S. (2012) "Plasmodium falciparum Neutral Aminopeptidases: Development of Novel Anti-Malarials by Understanding Enzyme Structure". in Proteinases as Drug Targets (Dunn, B. ed.), Royal Society Chemistry, Oxford. pp 169-185
Grigoryev, S., and McGowan, S. (2011) "Isolation and Characterisation of the Nuclear Serpin MENT". in Serpin Structure and Evolution (Whisstock, J., and Bird, P. eds.), Academic Press, UK. pp 29-48
Zhang, Q., Law, R., Buckle, A. M., Cabrita, L., McGowan, S., Irving, J. A., Faux N., Lesk, A. M., Bottomley, S. P., and Whisstock, J. C. (2007) "Serpins in Prokaryotes". in Molecular and Cellular Aspects of the Serpinopathies and Disorders in Serpin Activity. (Silverman, G., and Lomas, D. eds.), World Scientific, Imperial College Press, UK. Pp
Cheung, J. K., McGowan, S., and Rood, J. I. (2005) "Two-component signal transductions systems of the clostridia". in Handbook of the clostridia (Durre, P. ed.), CRC Press, USA. pp
McGowan, S. (2013) Three assays in one well: Antimalarial compound library screening using the FLUOstar Omega. (BMG Labtech, Application Note).
Whisstock, J. C., McGowan, S., Trenholme, K. R., Gardiner, D. L., and Dalton, J. P. (2009) Reply to Klemba: Intracellular processing of the membrane-bound PfA-M1 neutral aminopeptidase, a target for new antimalarials. Proc Natl Acad Sci U S A 106, E56. (Letter to the Editor, peer-reviewed).
The Structural Microbiology Laboratory is seeking enthusiastic students to join the team.
Exceptional graduate students with a high level (H1) Honours degree (or equivalent) in Biochemistry, Molecular Biology, Microbilogy or a related discipline are encouraged to apply. The scholarship is open to Australian and New Zealand citizens or permanent residents only and is funded at the rate of the Australian Postgraduate Award. Details of eligibility requirements to undertake the PhD are available at http://www.monash.edu.au/migr/research-degrees/
The scholarship offer is to the value of $25,392 per annum full-time rate (tax-free stipend)
To apply, please ensure you include with your application:
• a cover letter including a brief outline (1 page) detailing your interest in, and suitability for, the projects
• a detailed curriculum vitae including academic transcript(s)
• names and contact details of 2 academic referees.
Supporting a diverse workforce
National Health and Medical Research Council of Australia
Australian Research Council
Victorian Life Sciences Computation Initiative